The anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK) is altered by chromosomal rearrangements in non- Hodgkin's lymphoma (NHL) and the sarcoma known as inflammatory myofibroblastic tumor (IMT) to produce constitutively active ALK fusion proteins. A direct causal relationship exists between the expression of ALK fusions and the genesis of NHL or IMT. Conversely, inhibition of ALK activity reverses the malignant phenotype experimentally. The normal functions of ALK are incompletely known, and Alk knockout mice exhibit no gross or histopathologic abnormalities. Studies of ALK signal transduction have identified several previously known kinase substrates as important in mediating ALK signaling, but the complete spectrum of substrates required for normal or oncogenic signaling by the kinase is not yet clear. Given the excellent validation of ALK as a target for directed therapies, Aim 1a of this proposal seeks to develop ATP-competitive small molecule inhibitors of ALK and to determine their specificity, toxicity and efficacy in preclinical tumor models. To provide guidance for the rational, structure-based design of ALK inhibitors, the studies in Aim 1b will determine the X-ray crystallographic structure of the apo, inactive and tris-phosphorylated, fully active ALK KD, as well as the structure of the ALK KD co- crystallized with selected inhibitors. The RTK known as leukocyte tyrosine kinase (LTK) and ALK share remarkable homology and a partially overlapping expression pattern, suggesting possible functional redundancy. To better understand the function of these kinases in growth and development and assess the consequences of their simultaneous inhibition (as may occur pharmacologically even with highly specific ATP-competitive ALK inhibitors), mice that lack both Alk and Ltk will be generated and characterized in Aim 1c. Recent ALK signaling studies have identified a novel substrate, ALK-interacting nuclear protein 3 (ANP-3), that possesses the ability to alter both apoptosis and cell cycle transit, especially mitotic entry and exit. The studies in Aim 2a will seek to determine the exact role of ANP-3 in the normal regulation of apoptotic death and the cell cycle, focusing especially on M-phase transit, and to understand the mechanism and consequences of ANP-3 deregulation by oncogenic ALK fusions. Finally, in Aim 2b, the phenotypic effects of Anp-3 inactivation in vivo on embryonic development, cell cycle integrity and apoptosis control will be assessed. These studies will improve basic understanding of normal ALK functions and the mechanisms employed by ALK fusion proteins in malignancy, while also identifying small molecule ALK inhibitors for use as basic research tools and ultimately perhaps for therapeutic applications.